Non-thermal dielectric barrier discharge plasma (NT-Plasma) is a relatively new physics-based technology. Although there are few reports concerning the application of this technology to biological sciences, it is known that NT-plasma influences cell function mainly through activation of reactive oxygen and nitrogen species (ROS/RNS) signaling pathways. In collaboration with the Drexel Plasma Institute, arguably the foremost experts in the field of plasma physics in the United States, we propose to use NT-plasma as a tool to specifically manipulate cellular redox to promote MSC commitment and differentiation. The proposed study is based on recent observations that the NT-Plasma system promotes reactive oxygen species generation enhances development of embryonic structures and initiates the expression of many genes linked to cell differentiation. The first Specific Aim is to delineate the mechanism by which NT-Plasma generated ROS/RNS promotes MSC proliferation, commitment and differentiation; while simultaneously developing the NT-Plasma device for this application. We will test the hypothesis that NT-Plasma enhances stem cell differentiation along chondrogenic, endothelial and osteogenic lineages. Moreover, that this effect is mediated via ROS/RNS dependent signaling pathways that serve to influence the cell's oxidative state. To test this hypothesis, first, we will define the conditions that permit NT-Plasma to regulate the oxidative state of the cell. At the same time, we will fine tune the NT-Plasma system modulating the length of treatment, times of treatment and amplitude and discharge parameters. We will measure ROS and RNS, the redox status of the cells, the expression and activity of antioxidant proteins, as well as responsive signaling pathways. The second Specific Aim is to determine how NT-Plasma advances differentiation of progenitor cells in two model systems, an endochondral ossification system and a tissue engineered vascular tissue allograft. We propose to test the hypothesis that NT-Plasma positively influences stem cell commitment and differentiation in vivo. Each system provides a unique opportunity to evaluate the potential and feasibility of NT plasma treatment to enhance tissue healing and replacement, while at the same time gaining valuable understanding of the resulting signaling networks. If the goals of this application are successfully achieved, then the knowledge gained in development of NT-Plasma technology will be of transformative scientific and clinical importance. NT-Plasma's ability to amplify stem cell function will be an invaluable tool for tissue engineering and regenerative medicine in general, and will provide new insights into the basic biology of ROS signaling in stem cell biology.